Btk蛋白在红壤胶体上的吸附解吸行为及其对杀虫活性的影响
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国家自然科学基金项目(30170032)、天津市自然科学基金重点项目(08JCZDJC18800)、天津市农业科技成果转化项目(08ZHNZNC03300)联合资助


Adsorption and desorption of Btk proteins on red soil colloids and their insecticidal activity of conjunct Btk proteins
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    摘要:

    研究了苏云金芽胞杆菌库斯塔克亚种(Btk)两种分子量不同的杀虫蛋白(65 kDa和130 kDa)在红壤胶体上的吸附、解吸和杀虫活性。Btk杀虫蛋白容易被红壤胶体吸附,0.5 h即达到吸附平衡,65 kDa蛋白的吸附速率高于130 kDa蛋白。两种Btk杀虫蛋白在0.1 mol L-1 Tris缓冲液(pH8)中的等温吸附曲线均符合Langmuir方程(R2 >0.992),65 kDa蛋白的吸附量高于130 kDa蛋白。在10~50℃范围内,温度升高吸附量略有降低。pH 6~8范围内,吸附量随pH升高而下降。两种Bt蛋白在红壤胶体吸附前后对棉铃虫均具有杀虫活性,而且吸附后杀虫活性提高了2~5倍。解吸实验表明,Btk杀虫蛋白与红壤胶体结合比较牢固,吸附态65 kDa蛋白和130 kDa蛋白经3次去离子水解吸,总解吸率分别为37.2%和22.9%,经3次pH8的Tris 缓冲液解吸,总解吸率分别为20.7%和14.8%。红外光谱分析表明,两种分子量的Bt蛋白在吸附前后结构没有明显变化。透射电镜分析表明,吸附前后红壤胶体颗粒粒径未发生改变。吸附态Bt蛋白杀虫活性提高且不易被解吸,提示转Bt基因作物释放蛋白可能存在环境风险.

    Abstract:

    Adsorption, desorption and insecticidal activity of 65 kDa and 130 kDa proteins from Bacillus thuringiensis subsp. Kurstaki (Btk) on red soil colloids were studied. The proteins from Btk were easily adsorbed on red soil colloids and the adsorption reached equilibrium within 0.5 h The adsorption rate of 65 kDa protein was higher than that of 130 kDa protein The adsorption isotherms of the two proteins in Tris buffer (pH 8) followed the Langmuir equation (R2 > 0.992) and more 65 kDa proteins adsorbed than 130 kDa proteins. The adsorption rates of the two proteins decreased slightly with the rising temperature from 10�C to 50�C, and declined, too, with the rising pH value from 6 to 8 (Tris buffer). Both free and adsorbed proteins were toxic to the larvae of Heliothis armigera, and the insecticidal activity of adsorbed proteins was 2~5 times higher than that of free proteins. Desorption experiments revealed that the proteins were tightly bound onto the clays, and only 37.2% and 22.9% of the proteins, 65 kDa and 130 kDa, respectively, adsorbed at equilibrium was desorbed after three washes with deionised water, 20.7% and 14.8% was desorbed after three washes with Tris buffer (pH 8). Infrared absorption spectra showed that the proteins did not alter much in structure, either adsorbed or desorbed. The analysis using transmission electron microscopy (TEM) showed that no obvious changes was observed in particle size of soil colloids In red soil either before or after adsorption. Adsorbed Bt proteins were higher in insecticidal activity, but low in desorption rate, which means that release of proteins from transgenic Bt plants may pose a potential environmental risk.

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周学永,高建保,蔡鹏,黄巧云,喻子牛. Btk蛋白在红壤胶体上的吸附解吸行为及其对杀虫活性的影响[J].土壤学报,2009,46(3):480-487. DOI:10.11766/trxb200708100315 Zhou Xueyong, Gao Jianbao, Cai Peng, Huang Qiaoyun, Yu Ziniu. Adsorption and desorption of Btk proteins on red soil colloids and their insecticidal activity of conjunct Btk proteins[J]. Acta Pedologica Sinica,2009,46(3):480-487.

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